HIV/AIDS is a global pandemic with 37 million individuals living with HIV infection and approximately 40 million have died from AIDS worldwide. The objectives of this project are to define the unique epidemiological, clinical, virologic, and immunologic features of HIV and its co-infections in developing countries, to determine the viral kinetics associated with sexual transmission, and to characterize the different molecular strains of HIV for infectiousness and progression of disease. We continued our research on the development, validation and application of accurate HIV cross-sectional incidence testing to determine the impact of population level interventions, identification of populations for intervention trials and epidemic surveillance. HIV-1 subtype C infects >50% of all infected individuals, and we developed an accurate multi-assay algorithm (MAA) optimized for subtype C that uses two serologic markers and viral load. This MAA had a mean window period of 248 days (95% CI: 218, 284), a shadow of 306 days (95% CI: 255, 359), and provided the most accurate and precise incidence estimate when compared to the observed incidence in HPTN 068. We determined that the impact of early treatment is even more important than previously determined. When reevaluating the START trial, we identified individuals who were recently infected at the initiation of the trial. Immunologic benefits were far greater in these individuals compared to those who were not recently infected at the beginning of the trial. Using a phage display assay, we characterized antibody binding to >3,300 HIV peptides in 57 subtype C infected women from 2 weeks to 8 years post infection. We found that the number of unique epitopes targeted (antibody breadth) increased early in infection and then stabilizes or declines. A decline in antibody breadth 9 months to 2 years after infection was associated more rapid disease progression. We identify 266 peptides with increasing antibody reactivity over time and 43 peptides with decreasing reactivity over time. These data were used to design a prototype four-peptide serosignature to predict duration of HIV infection. We also demonstrated that epitope engineering can be used to optimize peptide binding properties for applications such as cross-sectional HIV incidence estimation. The limiting-antigen avidity (LAg-Avidity) assay with viral load (VL) >1,000 copies/mL is being used to estimate population-level HIV incidence in Africa. However, this has not been validated in East Africa where HIV-1 subtypes A and D circulate. Sera from persons seen in two surveys (2008-2009 and 2012-2013) limited to those who attended the previous round of the Rakai Community Cohort in Uganda were analyzed. The performance of the current LAg-Avidity protocol, with a mean duration of recent infection (MDRI) of 130 days and false recent rate (FRR) of 0%, was compared with subtype-specific MDRI and FRR, adjusted to subtype distributions. The observed incidence was 1.05/100 person years (py) 95% confidence interval (CI) 0.90-1.23 in 2008-2009 and 0.66/100py (95% CI 0.52-0.83) in 2012-2013. In contrast, the per-protocol LAg-Avidity incidence estimates were 1.63/100py (95% CI 0.97-2.30) in 2008-2009 and 2.55/100py (95% CI 1.51-3.59) in 2012-2013 (a significant increase, p<.05.) However, using a subtype-specific MDRI and FRR, the subtype adjusted incidence was 0.88% (95% CI 0.44-1.33) in 2008-2009 and 0.67% (95% CI 0.00-1.68) in 2012-2013, approximating the observed incidence trends. In this subtype A/D epidemic, the per protocol LAg-Avidity + VL assay overestimated HIV incidence and failed to detect declines in incidence. Adjustment for FRR, MDRI, and subtype distribution provided incidence estimates similar to empirically observed incidence level and trends. Thus, use of the LAg-Avidity assay in an A/D epidemic requires adjustment for subtype. The major barrier to curing HIV infection is the persistence of HIV in latently infected resting memory CD4+ T cells. Previous work from our section and the ICER Uganda team found that the latent viral reservoir (LVR) in our Ugandan population is over 3-fold smaller than that of a previously reported American population. This represents the first quantification of latently infected resting CD4+ T cells with replication competent virus in an ART treated, virally suppressed sub-Saharan African population. We expanded on this work to design and validate a novel Bayesian model for estimating the size of the latent viral reservoir that considers the clonality of the viral outgrowth populations. This new model significantly increases the accuracy of the estimate of the infectious units per million cells (IUPM) as compared to traditional methods. It also allows for the measurement of IUPMs for individual clonal variants allowing for the longitudinal measurement of these variants overtime. We have expanded our studies examining HIV superinfection and its effects on the pandemic by exploring the role of anti-HIV neutralizing antibodies (NAb) in the context of HIV superinfection in three studies. In two collaborative projects with researchers from the MRC-Uganda and the Danish HIV cohort we explored the level of NAb in individuals prior to HIV superinfection. The initial study examined a cohort of professional sex-workers and found that HIV superinfection can occur even in the presence of detectable NAb, whereas our work in Denmark found very little NAb response prior to HIV superinfection in a small number of cases. Our third project was the first study to identify a linked heterosexual HIV superinfection event where the superinfecting partner was known. In collaboration with Dr. John Mascola and Dr. Nicole Doria-Rose from the NIAID VRC, we generated fully infectious envelope clones from this couple and examined their NAb response to each others viruses before and after HIV superinfection. In this study we found that although the female partner possessed a moderate NAb response prior to superinfection, she had no detectable response to her husbands superinfecting strain indicating that a successful HIV vaccine will need to elicit a broad NAb response to reach maximum efficacy. South Africa has the largest HIV epidemic in the world, with 19% of the global number of people living with HIV, 15% of new infections and 11% of AIDS-related deaths. In a prospective study, we screened 1714 patients in an emergency department in Eastern Cape of South Africa for HIV. Four hundred (21.6%) patients were HIV positive, including 115 (6.2%) with newly diagnosed HIV infection. The overall prevalence of HIV infection was twice as high in females (29.8%) compared to males (15.4%). These studies will be expanded in other cities identify the scope of the epidemic there and to better define the clinical cascade of care and incidence. We have also continued our work examining HIV positive to HIV positive organ transplants in South Africa and the US. As part of this later collaboration we examined the benefit of giving organs from donors that are found to be false-positive for HIV during the organ procurement process to HIV+ recipients as allowed under the HOPE act. Using data collected during our Pilot study we estimate that allowing these organs to be used will increase the overall donor pool by 50-100 donors a year.